The purpose of the current study was to determine the effects

The purpose of the current study was to determine the effects of the introduction of polysaccharide chains onto the molecular surface of buckwheat proteins on buckwheat protein surface functionality. the antigenicity of WBP. Moench) flour was obtained from the Education and Research Center of Alpine Field Science in Shinshu University (Ina Nagano Japan). Dextrans with five different molecular masses 6 kDa (DX6) 15 kDa (DX17.5) 40 kDa (DX40) 50 kDa (DX70) and 200 kDa (DX200) were purchased from MRC Polysaccharide Co. Inc. (Toyama Japan). Human sera were obtained from four buckwheat-allergy subjects (one Clec1b male and three females; 12 to 45 years old; GSK1904529A Table 1). The Sephacryl S-300 column and Q Sepharose FF were obtained from GE Healthcare (Tokyo Japan). Goat anti-human IgE labeled with HRP was obtained from MorphoSys UK Ltd. (Oxford UK). All other reagents were of biochemical grade. Table 1 Characteristics of human sera used in this study Preparation of whole buckwheat protein fraction The WBP was prepared according to Urisu et al. (8) with some modifications. Common buckwheat flour was defatted using acetone and air-dried for one hour to completely remove the solvent. The resulting powder was dissolved in distilled water 50 mM phosphate buffer pH 7.5 (PBS) PBS made up of 0.1 M NaCl or PBS containing 0.5 M NaCl and stirred overnight at 4°C. The extract was centrifuged at 6 0 for 15 min at 4°C and the supernatant was filtered through No. 5A filter paper once (Advantec Co. Ltd. Tokyo Japan). The proteins were then precipitated out of the answer by stirring overnight at 4°C with 80% saturated ammonium sulfate. The resulting precipitate was collected by centrifugation dialyzed against distilled water lyophilized and used as WBP powder. Preparation of WBP-dextran conjugates Maillard-type glycation was used to prepare WBP-dextran conjugates according to the method described by Kato et al. (17). Briefly WBP was mixed 1:1 with DX6 1 with DX17.5 1 with DX40 1 with DX70 or 1:30 with DX200. These mixtures were incubated at 60°C and 79% relative humidity for 2 weeks. The resulting GSK1904529A WBP-dextran conjugates were separated from free proteins and carbohydrates using size exclusion chromatography with a Sephacryl S-300 column followed by ion exchange chromatography with Q Sepharose FF and GSK1904529A used for further experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was conducted according to the method described Laemmli (19) using a 15% (w/w) acrylamide separating gel with a 5% (w/w) stacking gel made up of 1% (w/v) SDS. Samples were heated at 100°C for 5 min in Tris-glycine buffer (pH 8.8) containing 1% SDS and 1% (v/v) 2-mercaptoethanol. Electrophoresis was carried out at a GSK1904529A constant current of 15 mA for 3 h using an electrophoretic buffer of Tris-glycine made up of 0.1% SDS. After electrophoresis the gels were stained for protein and carbohydrate with 0.025% (w/v) Coomassie brilliant blue R-250 solution and 0.5% (w/v) periodic acid-Fuchsin solution (20) respectively. Determination of free amino groups 2 4 6 sulfonic acid (TNBS) was used to quantify the free amino groups present in the WBP-dextran conjugates according to the method of Haynes et al. (21). Determination of solubility The solubility of WBP in distilled water PBS PBS made up of 0.1 M NaCl or PBS containing 0.5 M NaCl was assessed by measuring the absorbance (at 280 nm) of the supernatants of 1 1 mg protein/mL solutions after centrifugation at 6 0 for 20 min at 4°C. Measurement of emulsifying properties The emulsifying properties of WBP were determined according to the method described by Pearce and Kinsella (22). Samples were dissolved in PBS at a concentration of 0.1% and 3 mL of the sample answer was homogenized with 1.0 mL of corn oil using a Polytron PT3100 (Kinematica AG Luzern Switzerland) homogenizer at 6 0 for 1 min at 20°C to prepare an O/W-type emulsion. One-hundred microliter aliquots of the emulsion were taken from the bottom of the test tube after standing for 0 min 1 min 3 min 5 min 10 min and 20 min. Each aliquot was diluted with 5.0 mL of 0.1% SDS answer. The absorbance of the diluted emulsions was measured at 500 GSK1904529A nm. The relative emulsifying activity of each sample is presented as the absorbance measured at 500 nm immediately after emulsion formation. The emulsion stability was estimated by measuring the half-life time for emulsion decay while standing for 20 min. Immuno-dot blotting assay A nitrocellulose membrane sheet was soaked.